Dynamics of nasopharyngeal tract phageome and association with disease severity and age of patients during three waves of COVID‐19

Abstract In December 2019, several patients were hospitalized and diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, which subsequently led to a global pandemic. To date, there are no studies evaluating the relationship between the respiratory phageome and the SARS‐CoV‐2 infection. The current study investigated the phageome profiles in the nasopharyngeal swabs collected from 55 patients during the three different waves of coronavirus disease 2019 (COVID‐19) in the Campania Region (Southern Italy). Data obtained from the taxonomic profiling show that phage families belonging to the order Caudovirales have a high abundance in the patient samples. Moreover, the severity of the COVID‐19 infection seems to be correlated with the phage abundance.

Indeed, Bacteroides phages and phages belonging to the Siphoviridae, Myoviridae, and Podoviridae families were known to be abundant in fecal samples of patients with Crohn's disease, explaining the high Bacteroides burden in the intestines of these patients. 13  September-November 2020); and the third group (collection date: January-February 2021). In total, 25 samples belonged to the first, as well as the second groups and 5 to the third. Thirty-one percent of patients were female (n = 17), and 65% were male (n = 36), with a median age of 59 years, ranging from 8 to 91 years. The study was approved by the Ethics Committee of "Campania Sud" (approval code: 206/2021) and was conducted according to the Declaration of Helsinki. Samples were also divided based on the severity of symptoms and were clustered into nonsevere (n = 39), moderate (n = 6), and severe (n = 10) as described in Ferravante et al. 14   were imported in the HOME-BIO pipeline. 15 The "Quality control" module was set to remove low-quality reads and host-related sequences, filtering out reads that mapped on the human reference genome (GRCh38.p13 release 37). Phage taxonomy profiling was obtained by querying RefSeq complete viral genomes/proteins database. Classification reports were then processed in R software (version 3.6.3) and normalized in reads per million (RPM) values (RPM mapped on the viral database). The raw read number obtained with the HOME-BIO pipeline was normalized in million mapped reads on the viral database. This normalization criterion was chosen to avoid problems related to different sequencing depths and to highlight all the small differences in terms of read abundance, as described in Di Gaspero et al. 16 Phage families identified by less than three reads in at least 50% of the analyzed samples were filtered out from the analysis.
Then, the differential distribution of phage families was computed as a ratio between the mean values of RPM in the groups analyzed.
Statistical significance was computed by applying a T-test followed by Bonferroni correction. Only comparisons with a p value ≤ 0.05 were considered significant, as described in Giugliano et al. 8 3 Overall, the phage abundance has the highest RPM values in the I wave, and the RPM values decreased in waves II and/or III ( Figure 1A).
The reasons behind this decrease may be explained by the restrictions such as wearing masks, reducing human contact, maintaining distance, and even strict curfews applied around the world during the pandemic.
While Microviridae belongs to the order Petitvirales, all others Siphoviridae were found to be associated with T2D. 25

| CONCLUSION
In the presented study, an analysis of nasopharyngeal swabs was performed to taxonomically classify the bacteriophage population in the patients from the Campania region infected with SARS-CoV-2.
F I G U R E 1 Distribution of RPM values related to detected phage families among the three time periods (A), disease severity degrees (B) and age quartiles (C). The significant comparisons between analyzed groups, associated with a p-value ≤ 0.05, are indicated with an asterisk (*). FERRAVANTE ET AL.